ABZYMES/
CATALYTIC ANTIBODIES
20P04
Introduction
What is an ‘Antibody (Ab)’?
• Antibodies are the recognition arm of the immune system that are
activated when the body is infected with a bacterium or virus.
• Antibody molecules are produced by the immune
system to bind and neutralize foreign substances
called antigens.
• Foreign proteins of bacteria and viruses, as well as
some small chemical molecules called haptens,
act as antigens, and elicit the production of antibodies
to protect the host from harm
ABZYMES- derived from Antibody ‘Ab’ and enzymes ‘zymes’
• Antibody binds to its own unique target similar to a key fitting in a
lock. Resembling the lock and key binding mechanism of enzyme and
substrate.
• Abzymes are not full fledged enzymes–they are ENZYME MIMICS
and form a new class of biocatalysts.
• They are produced from monoclonal antibodies and due to their
catalytic feature – catalytic monoclonal Ab
Keynotes
• Haptens are small molecules that elicit an immune response only
when attached to a large carrier such as a protein; the carrier may be
one that also does not elicit an immune response by itself.
• Transition state is a high-energy state, is unstable, reactant quickly
proceed to the next step of the chemical reaction and do not linger in
this state.
• Small-molecule haptens must be attached to a large protein molecule,
called a carrier protein.
• Antibodies that are produced after immunization with the hapten-
carrier protein conjugate are complementary to, and thus specifically
bind, the hapten.
The human body is capable of producing antibodies to virtually any
encountered antigen.
The main focus of ABZYME development:
The body can generate Ab to every Antigen (Ag) in our body
We should be able to generate Ab to a hapten (Ag equivalent) which
represents the “transition state” of the given organic reaction.
Leads to acceleration/ catalysis of the reaction.
• Antibody molecules simply bind; they do not catalyze reactions.
However, catalytic antibodies are produced when animals are
immunized with hapten molecules that are specially designed to elicit
antibodies that have binding pockets capable of catalyzing chemical
reactions.
Binding forces within the antibody binding pocket
Stabilizes transition states and intermediates
Lowers the reaction's energy barrier, therefore increasing the reaction
rate
How does this occur?
• The Ab has a binding site complementary to a transition state in terms
of both 3-D geometry and charge distribution.
• This complementarity leads to catalysis by encouraging the substrate
to adopt a transition state-like geometry and charge distribution.
This leads to:
- Lowered energy barrier
- Prevention of unwanted product formation thereby, increasing reaction
selectivity.
• Production of Ab with binding pockets complementary to transition
states is a tedious task. Why?
Due to the sole fact that true transition states and most reaction
intermediates are unstable.
• Since true transition states or intermediates cannot be isolated or used
as haptens for immunization, what can be done??
This guides us to use of so called ‘Transition State Analogues (TSA)’
What are TSA molecules?
TSA:
- Stable molecules that simply resemble a transition state (or
intermediate) for a specific reaction w.r.t geometry and charge
distribution.
- It resembles a true reaction transition state or intermediate to a point
that, the formed antibodies will also be complementary to that
transition state or intermediate and thus lead to the catalysis of the
reaction.
• It is unclear if Ab endowed with catalytic activity are an intrinsic part
of the physiological immune system or if the catalytic function of Ab
is gained solely after proper stimulation of the immune system by an
appropriate immunogen.
• Catalytic antibodies are generated spontaneously by the immune
system in the absence of immunization by an exogenous peptide or a
protein.
• Catalytic IgG and IgM have been documented in the serum of healthy
individuals while antibodies with protein kinase and DNase activities
have been detected in the milk of healthy mothers.
• Natural abzymes can also be found in patients with autoimmune
diseases such as systemic lupus erythematosus (SLE).
History
• The basis for the production of catalytic antibodies is in Pauling's
lecture on the action of enzymes wherein he stated that enzymes are
complementary to the structures of the transition states of the reactions
that they catalyze
• The possibility of catalyzing a reaction by means of an Ab which
binds the transition state was first suggested by William P. Jencks in
1969.
• In 1994 Peter G. Schultz and Richard A. Lerner received the
prestigious Wolf Prize in Chemistry for developing catalytic
antibodies for many reactions and popularizing their study into a
significant sub-field of enzymology.
Basis for its functioning
• Enzymes function by lowering the activation energy of the transition
state of a chemical reaction.
• If an Ab is developed to bind to a molecule that is structurally and
electronically similar to the transition state of a given chemical
reaction, the developed Ab will:
bind stabilizes the transition state (just like a natural enzyme)
lowering the activation energy of the reaction, thus catalyzing the
reaction.
To generate abzymes, one must:
- design and synthesize the transition state analog for a desired reaction.
The problem is to synthesize the transition state analog, but this may be
solved through the cooperation with organic chemistry.
Hybridoma technology
• Used for the generation of monoclonal Ab.
• The direct screening of culture supernatants for
Ab catalysis was not possible due to:
- low activity of the abzymes
- low abundancy is also a problem.
- The catalytic activity can be examined only after the monoclonal
antibodies has been obtained.
- It is not a rare case not to obtain even one monoclonal abzyme after
preparing a number of monoclonal antibodies, this leads to increase
costs.
Modifications such as the non-hybridoma methodologies, using
combinatorial variable-region cloning in phages enables us to select
abzymes from among millions of possibilities.
• Catalytic activity detection in the early stage of cloning is carried out
by a screening method termed catELISA.
Here,
- Substrates attached to BSA are immobilized on ELISA plates
- Plates are incubated with anti-hapten (TSA) antibodies, which may
contain abzymes to catalyze the immobilized substrates to the products
via the transition states.
- Then the products on the plates are incubated with rabbit serum containing
anti-product antibodies.
- The rabbit antibodies bound to the immobilized products can be detected
using the enzyme-labeled anti-rabbit IgG antibodies
Polyclonal Catalytic antibodies
The abzymes first generated were monoclonal antibodies and early
attempts to produce polyclonal catalytic antibodies failed.
- Human autoantibodies catalyzing hydrolytic reactions have been
reported.
The advantages of polyclonal abzymes may be as follows:
(i). The relative simplicity, rapidity, and low cost of producing
polyclonal as against monoclonal antibodies
(ii). Their use as catalysts for technological applications
(iii). Their use for the assessment of potential therapeutic strategies involving
catalytic activity induced in the serum
(iv). For hapten design by analyzing sets of structurally related haptens and
comparing catalytic activities of the resulting immune response, as measured
with polyclonal antibodies
It is recommended to test the feasibility of the design and synthesis of
haptens by generating polyclonal abzymes before trying to obtain
monoclonal ones.
• To date abzymes display only weak, modest catalytic activity.
• This has led to the engineering of some abzymes with metal ions and
other cofactors to improve their catalytic activity.
• Studies on abzymes has yielded important insights into reaction
mechanisms, enzyme structure and function, catalysis and the immune
system itself
References
• Catalytic antibodies (abzymes): From concept to application Department of
Pure & Applied Chemistry, WestCHEM, University of Strathclyde, Thomas
Graham Building, 295 Cathedral Street, Glasgow G1 1XL, Scotland, UK
• Recent Advances in Abzyme Studies, Haruo Suzuki Department of
Biophysical Chemistry, Kitasato University School of Medicine,
Saganuhara, Kanagawa 228, Received for publication, November 2, 1993
• [Link]
bzyme
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